1. Field of the Invention
The present invention relates to a method of establishing an EBV-infected stomach cancer cell line from stomach cancer tissues infected with EBV, as well as EBV infecting cultured epithelial cells.
2. Description of the Related Art
EBV (Epstein-Barr Virus) is a DNA virus belonging to the family of human herpes virus. When adult persons are first infected with it, infectious mononucleosis (TM) occurs, but in Japan, the majority of persons are first infected latently with the virus at the infant stage, and through life, latent infection continues. Accordingly, this virus has been suspected to be that causing many diseases including various cancers and autoimmune diseases, but there are many features unrevealed except that the connection with Burkitt lymphoma in Africa (Epstein M A, Barr Y M, Lancet Vol. 1:252-253, 1964) and upper pharyngeal cancer in the southern part of China (Pathnabathan R. et al., New Engl. J. Med. 333(11):693-698, 1995) was proven.
In recent years, an EBV gene was detected in stomach cancer cells (Shousha S. et al., J. Clin. Pathol. 47:695-698, 1994) and further the EBV gene was found to be monoclonal (Imai S. et al., Proc. Natl. Acad. Sci. USA, 91:1931-1935, 1994), and since it was suggested that the EBV gene may be involved in the canceration process, it came to be thought that EBV is involved in at least a part of stomach cancers.
At present, no diagnostic method has been established for specifying whether EBV is involved in canceration in each patient with stomach cancer, so it is not known whether the stomach cancer caused by EBV, as compared with other stomach cancers, has distinct characters such as ease of metastasis, rapidness of the progress, drug sensitivity etc. While establishment of a diagnostic method therefor is desired, establishment of a therapeutic method and particularly development of chemotherapy for stomach cancer in which EBV is involved are important tasks. For establishment of a diagnostic method and development of a chemotherapy, establishment of a stomach cancer cell line cancerated by EBV is essential.
Recently, the present inventors reported establishment of epithelial cells GT38 and GT39 derived from stomach normal tissues in patients with stomach cancer (Tajima M. et al., Jpn. J. Cancer Res. 89:262-268, 1998). It was surprising that EBV whose natural host is lymphoid B cells infects epithelial cells to produce EBV-related antigens though in a small amount, but for establishment of the diagnostic method and development of the chemotherapy, it was desired to establish a stomach cancer cell line which was established not from normal tissues but from cancer tissues, that is, a stomach cancer cell line which certainly underwent in vivo canceration.
Further, if canceration occurs due to a product of the EBV gene, a new type of anticancer drug inhibiting the mechanism of canceration can be developed. To analyze the mechanism of canceration of stomach cancer by EBV, it would be necessary to infect epithelial cells with EBV in order to analyze which gene in EBV is involved in the canceration.
However, it was not possible to efficiently infect cultured epithelial cells with EBV in vitro, although the relationship between EBV and the stomach cancer was believed, since the EBV gene was also found in vivo in epithelial cells of stomach cancer cells etc. (Shousha S. et al., J. Olin. Pathol. 47:695-698, 1994). Up to now, it is has been reported that genetic recombinant EBV was prepared for infecting cultured epithelial cells with EBV (Borza CM et al., J. Virol. 72(9):7577-82, 1998; Imai S. et al., J. Virol. 72(5):4381-8, 1998). In these reports, a method of amplifying and capturing a very rare phenomenon, that is, a method of infecting the cells with EBV having a drug resistance gene (Neor) inserted into it followed by selection with the drug (neomycin), was used. For example, in the report of Imai in 1998, it was reported that upon infection of a culture supernatant (400,000 cells), 32 cells at the maximum were successfully infected, and there is no guarantee that such a rare phenomenon reflects naturally occurring infection with EBV. Up to now, there is no report on efficient infection of epithelial cells with EBV whose gene is not subjected to recombination, but a system for infection of epithelial cells with EBV whose gene is not modified is necessary to analyze the mechanism of actually occurring in vivo canceration by EBV.
In an epidemiological study, it was found that the antibody titer toward EBV-related antigens i.e. viral capsid antigen (VCA) and latent membrane protein (LMP) in patients with stomach cancer is higher than in healthy persons (Masako Tajima, xe2x80x9cRinsho To Virusxe2x80x9d (Clinics and Virus), Vol. 25, No. 3:169-176, 1997), and the relationship between the stomach cancer and EBV was thus supported, and simultaneously the worth of diagnosis of the antibody titer toward EBV-related antigens came to attract attention.
To utilize the EBV-related antigens in diagnosis of stomach cancer, a stable supply of EBV-related antigens is essential. In consideration of posttranslational modification of proteins, cells producing EBV-related antigens are desirably epithelial cells, particularly stomach cancer cells. However, generally known EBV-infected cells such as B95-8 and Daudi are lymphoid cells and have the problem of low production of VCA and LMP. Further, GT38 and GT39 established from normal tissues in patients with stomach cancer, reported recently by the present inventors (Tajima M. et al., Jpn. J. Cancer Res. 89:262-268, 1998), had the problem of low production of VCA and LMP, as well. Further, depending on culture conditions, differentiation and induction occurred in these cells, so there was the problem of unstable production of EBV-related antigens. It was desired that a stomach cancer cell line stably producing EBV-related antigens is established from stomach cancer tissues considered to have stable traits at the final stage of canceration.
The object of the present invention is to establish an EBV-infected stomach cancer cell line for stable production of EBV-related antigens used in screening of a chemotherapeutic agent and a diagnostic agent and to establish an EBV strain efficiently infecting epithelial cells, in order to develop a diagnostic method and chemotherapy for stomach cancer cancerated by EBV.
For screening of a chemotherapeutic agent for stomach cancer cancerated by EBV and for establishing a cell line for stable production of EBV-related antigens, the present inventors speculated that a cell line can be established from stomach cancer tissues reflecting in vivo canceration and having stable traits as cancer.
Accordingly, stomach cancer tissues obtained by removing cancer lesions were treated with an antibiotic and then cultured in MEM medium mixed with an equal volume of keratinocyte-SFM and supplemented with 10% inactivated fetal calf serum and as a result of their eager study, the present inventors succeeded in establishing the stomach cancer cell line GTC-4 to arrive at the present invention.
The established GTC-4 cells stably produce EBV-related antigens and causes formation of tumors in immunodeficient mice and further causes metastasis to lymph nodes, so the cells are applicable to in vitro and in vivo screening of an anticancer drug targeted at EBV-infected stomach cancer cells.
Further, upon infection of epithelial cells with a culture supernatant of GTC-4, expression of the EBV-related antigen VCA was observed while cytopathic effect (CPE) was brought about, and GTC-4 was thus confirmed to produce EBV infecting epithelial cells. The EBV can be used for analysis of the mechanism of canceration by EBV as well as for screening of a drug that inhibits the growth of cancer cells by inhibiting the mechanism of canceration.
Further, GTC-4 cells produce the EBV-related antigen stably in a large amount and thus can be used as cells for production of the EBV-related antigen for use in a diagnostic drug for stomach cancer cancerated by EBV and as cells for preparation of mRNA and cDNA coding for the EBV-related antigen in the case of production of the EBV-related antigen by genetic recombination means.
That is, the present invention relates to a method of establishing an EBV-infected stomach cancer cell line by use of EBV-infected stomach cancer tissues; an EBV-infected stomach cancer cell line obtained in the method; the EBV-infected stomach cancer cell line GTC-4; and a method of screening an anticancer agent by use of the EBV-infected stomach cancer cell line. Further, the present invention relates to EBV infecting epithelial cells; the EBV separated from epithelial cells; the EBV separated from GTC-4; a method of screening an antiviral agent by use of the EBV; and a method of screening an anticancer agent by use of epithelial cells infected with the EBV.
Further, the present invention relates to a diagnostic drug for stomach cancer by use of an EBV-related antigen obtained from the EBV-infected stomach cancer cell line; a method of producing an EBV-related antigen which comprises the step of culturing the EBV-infected stomach cancer cell line; and the EBV-related antigen obtained in the method. Further, the present invention relates to a method of producing mRNA and then cDNA coding for the EBV-related antigen from the EBV-infected stomach cancer cell line. In the present invention, the EBV-related antigen includes, but is not limited to, latent membrane protein (LMP), viral capsid antigen (VCA) and EBV nuclear antigen (EBNA).